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How plants adapt to freezing temperatures and acclimate to survive the formation of ice within their tissues has been a subject of study for botanists and plant scientists since the latter part of the 19th century. In recent years, there has been an explosion of information on this topic and molecular biology has provided new and exciting opportunities to better understand the genes involved in cold adaptation, freezing response and environmental stress in general. Despite an exponential increase in our understanding of freezing tolerance, understanding cold hardiness in a manner that allows one to actually improve this trait in economically important crops has proved to be an elusive goal. This is partly because of the growing recognition of the complexity of cold adaptation. The ability of plants to adapt to and survive freezing temperatures has many facets, which are often species specific, and are the result of the response to many environmental cues, rather than just low temperature. This is perhaps underappreciated in the design of many controlled environment experiments resulting in data that reflects the response to the experimental conditions but may not reflect actual mechanisms of cold hardiness in the field. The information and opinions presented in this report are an attempt to illustrate the many facets of cold hardiness, emphasize the importance of context in conducting cold hardiness research, and pose, in our view, a few of the critical questions that still need to be addressed.

ABSTlRACTThe effect of abscisic acid (ABA) on the cold hardiness of cell suspension was investigted. Cell (2,(15)(16)(17). Because of the involvement of ABA in many environmental stresses, ABA has been proposed as a common mediator for plant stress response (5). However, the role of ABA in the adaptation of plants to freezing stress is not clear. Irving and Lamphear (13) were among the first to suggest that ABA may be involved in the cold hardening process.Two lines of evidence support the involvement of ABA in plant cold hardening. First, an increase in the endogenous level of ABA has been observed when plants are exposed to low temperature (2,5,(14)(15)(16). Second, exogenous applications of ABA have been shown to increase the cold hardiness in certain plants (2,3,15,16). However, attempts to increase cold hardiness by exogenous applications of ABA have not always been successful (6, 9). In these studies ABA was applied either as a foliar spray (6) or through the roots in a hydroponics solution (10). The failure ofexogenously added ABA to increase cold hardiness in these studies may be due to the lack of uptake, or microbial and enzymic degradation. (20), and by regrowth.The procedures for measuring TTC reduction was essentially the same as that described by Towill and Mazur (18) with the following modifications: thawed cells (0.2 g fresh weight) were incubated in 3 ml TTC solution (0.08% TTC in 0.05 M sodium phosphate buffer, pH 7.4) for 18 h at 20°C in the dark. After TTC incubation, the cells were rinsed with 3 ml distilled H20 and the reduced TTC extracted with 5 ml of 95% ethanol. TTC reduction was determined by measuring A at 485 nm. Freezing injury is expressed as the percentage ofdecrease in TTC reduction as compared to a nonfrozen control. The temperature which resulted in a 50% decrease TTC reduction is defined as the 50% killing temperature (LT50).Viability as determined by regrowth was conducted under sterile conditions. Following the freeze test, cell aggregates approximately 1 mm in diameter were transferred to 0.7% agar containing the appropriate medium. After I month at 20°C in the dark, the growth of the callus was determined.

Infrared video thermography was used to observe ice nucleation temperatures, patterns of ice formation, and freezing rates in nonacclimated and cold acclimated leaves of a spring (cv Quest) and a winter (cv Express) canola (Brassica napus). Distinctly different freezing patterns were observed, and the effect of water content, sugars, and soluble proteins on the freezing process was characterized. When freezing was initiated at a warm subzero temperature, ice growth rapidly spread throughout nonacclimated leaves. In contrast, acclimated leaves initiated freezing in a horseshoe pattern beginning at the uppermost edge followed by a slow progression of ice formation across the leaf. However, when acclimated leaves, either previously killed by a slow freeze (2 degrees C h(-1)) or by direct submersion in liquid nitrogen, were refrozen their freezing pattern was similar to nonacclimated leaves. A novel technique was developed using filter paper strips to determine the effects of both sugars and proteins on the rate of freezing of cell extracts. Cell sap from nonacclimated leaves froze 3-fold faster than extracts from acclimated leaves. The rate of freezing in leaves was strongly dependent upon the osmotic potential of the leaves. Simple sugars had a much greater effect on freezing rate than proteins. Nonacclimated leaves containing high water content did not supercool as much as acclimated leaves. Additionally, wetted leaves did not supercool as much as nonwetted leaves. As expected, cell solutes depressed the nucleation temperature of leaves. The use of infrared thermography has revealed that the freezing process in plants is a complex process, reminding us that many aspects of freezing tolerance occur at a whole plant level involving aspects of plant structure and metabolites rather than just the expression of specific genes alone.

The freezing tolerance or cold acclimation of plants is enhanced over a period of time by temperatures below 10°C and by a short photoperiod in certain species of trees and grasses. During this process, freezing tolerance increases 2-8°C in spring annuals, 10-30°C in winter annuals, and 20-200°C in tree species. Gene upregulation and downregulation have been demonstrated to be involved in response to environmental cues such as low temperature. Evidence suggests ABA can substitute for the low temperature stimulus, provided there is also an adequate supply of sugars. Evidence also suggests there may be ABA-dependent and ABA-independent pathways involved in the acclimation process. This review summarizes the role of ABA in cold acclimation from both a historical and recent perspective. It is concluded that it is highly unlikely that ABA regulates all the genes associated with cold acclimation; however, it definitely regulates many of the genes associated with an increase in freezing tolerance.

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A simplified method for the isolation of a plasma membrane-enriched fraction from plants utilzing an aqueous two-polymer phase system Is outined. Mainly, the plant used was Orchard grass (Dacty1s g8wmera L.). The two-phase system consisted of 5.6% (w/w) of dextran T500 and 5.6% (w/w) of polyethyleneglycol 4000 in 0.5 molar sorbitol-IS miflor , and 30 millimolar NaCl. In this system, the plsm membranes and the other membranes were preferentally partitioned into the top phase and into the lower phase, respectively. The purity of the isolated plasma membrane was sufficiently high even after a singe partition (i.e about 85% purity) and more than 90% purity was obtained after repeating the partition in a newly prepared lower phase The plasma membrane was identified with the aid of phosphotungstic acid-chromic acid stain and the association of vanadate-sensitive Mge'-ATPase. The plasma membrane-associated ATPase had a pH optimum at 6.5 and showed a hih specificity for Mge and ATP. KCI stimulation was low (6% stimulation) at the pH optimum, but a relatively high stimulation (23%) occurred at pH 5.5. This method for plasma membrane isolation may be appicable to a wide variety of plants and plant tissue incluing green leaves.Recently, the plasma membrane has received much attention due to its role in cell wall biosynthesis (29), ion transport (18,22,40), hormone action (3, 11), phytochrome responses (34), disease resistance (41), and stress responses (24). In some cases, the role of the plasma membrane can be best studied in situ using isolated protoplasts; however, in many studies the plasma membrane has to be isolated from intact tissue. Both the purity and the quantity should be sufficient to do detailed studies. A plasma membraneenriched fraction has been isolated from roots (15,18,21,23,30,33,42,43) and protoplasts (7,13,32). In most cases, the method employed for the isolation requires mechanical disruption of cells followed by differential and density gradient centrifugations. One of the major problems encountered by investigators attempting to isolate the plasma membrane from differentiated tissues, especially green tissues, is the cross-contamination of fragmented subcellular organelles such as chloroplasts, nuclei tonoplasts, and mitochondria. Chloroplast fragments are very difficult to separate from other membranes on the basis of differences in sedimentation characteristics. In his recent review on plasma membrane isolation

Superoxide dismutase (SOD) gene expression was investigated to elucidate its role in drought and freezing tolerance in spring and winter wheat (Triticum aestivum). cDNAs encoding chloroplastic Cu/ZnSODs and mitochondrial MnSODs were isolated from wheat. MnSOD and Cu/ZnSOD genes were mapped to the long arms of the homologous group-2 and -7 chromosomes, respectively. Northern blots indicated that MnSOD genes were drought inducible and decreased after rehydration. In contrast, Cu/ZnSOD mRNA was not drought inducible but increased after rehydration. In both spring and winter wheat seedlings exposed to 2°C, MnSOD transcripts attained maximum levels between 7 and 49 d. Transcripts of Cu/ ZnSOD mRNA were detected sooner in winter than in spring wheat; however, they disappeared after 21 d of acclimation. Transcripts of both classes of SOD genes increased during natural acclimation in both spring and winter types. Exposure of fully hardened plants to three nonlethal freeze-thaw cycles resulted in Cu/Zn mRNA accumulation; however, MnSOD mRNA levels declined in spring wheat but remained unchanged in winter wheat. The results of the dehydration and freeze-thaw-cycle experiments suggest that winter wheat has evolved a more effective stress-repair mechanism than spring wheat.Active oxygen species such as superoxide, H 2 O 2 , and hydroxyl radicals are by-products of normal cell metabolism. These active oxygen species result in the peroxidation of membrane lipids (Mead, 1976), breakage of DNA strands (Brawn and Fridovich, 1981), and inactivation of enzymes (Fucci et al., 1983). The conditions leading to damage caused by active oxygen species are referred to as oxidative stress. Both chloroplasts and mitochondria can produce active oxygen species either under normal growth conditions or during exposure to various stresses. The PSI electron-transport chain contains a number of autooxidizable enzymes that reduce O 2 to superoxide (Badger, 1985; Asada and Takahashi, 1987; Asada, 1994), and evidence indicates that superoxide and H 2 O 2 can also be produced by PSII under high-light intensities (Landgraf et al., 1995).During mitochondrial respiration, reactive oxygen species are also generated via the reactions of the electrontransport chain (Rich and Bonner, 1978;Bowler et al., 1991).Active oxygen species are also generated during chemical and environmental stresses, including chilling and freezing (Wise and Naylor, 1987;Senaratna et al., 1988; Kendall and McKersie, 1989;Tsang et al., 1991;McKersie et al., 1993), drought (Perl-Treves and Galun, 1991; Price and Hendry, 1991), desiccation (Senaratna et al., 1985;Leprince et al., 1990), flooding (Hunter et al., 1983;Van Toai and Bolles, 1991), herbicide treatment (Malan et al., 1990; Kurepa et al., 1997), pathogen attack (Montalbini and Buonaurio, 1986; Buonaurio et al., 1987; Koch and Slusarenko, 1990), and ionizing radiation (Niwa et al., 1977).SODs are a group of metalloenzymes that protect cells from superoxide radicals by catalyzing the dismutation of the superoxide radical to...